IN CULTURE

IL T R A ST R U C T U R E O F C E L L S A N D T IS SU E S IN C U L T U R E

S y m p o s i a B i o l o g i c a H u n g a r i c a

ULTRASTRUCTURAL FEATURES OF CELLS AND TISSUES

IN CULTURE

edited by

I. TÖRŐ and GY. RAPPAY

(Symposia Biologica H ungariea 14) Tissue culturing has become one of the basic methods of present day experi­

m ental biology. I t is the purpose of the annual Conferences of the E uro­

pean Tissue Culture Society to promote the application of this method in the study of all kinds of cell-biological problems.

The papers in this volume are those of the first day of the 1971 meeting held in Budapest and all the contribu­

tions are dealing with the fine struc­

ture of cultured tissues and cells. The papers cover a wide range of experi­

mental work w ith objects like lympho- retieular cells, tum our cells, endocrine as well as plant cells. The results indi­

cate th a t irrespective of whether the original u ltrastructure is retained or lost in culture, various functions char­

acterizing the cells i n v iv o are still apparent under i n v itr o conditions.

This symposium has been a further proof of the im portance of the tissue culture method in studying the control of various vital functions on the cellu­

lar level. The electron micrographs presented are especially valuable to cytologists.

A K A D É M I A I K I A D Ó Publishing House o f the Hungarian Academy

of Sciences, Budapest

S y m p o s i a B i o l o g i c a H u n g a r i с a

1 4

R e d i g i t

I. T Ö R Ő e t G Y . R A P P A Y

Vol.

r

—P1828 1L. Я V ГТТ

A K A D É M IA I K IA D Ó , B U D A PEST 1972

U LT R A S T R U С T L R A L F E AT U R F S O F C E L L S

A N D T I S S U E S I N C U L T U R E

E d ite d b y

I. TÖRÖ

I ) e p a r tm e n t o f H is to lo g y a n d E m b ry o lo g y , S em m elw eis M ed ical U n iv e rs ity , B u d a p e s t

a n d

GY. RAP PA Y

I n s t i t u t e o f E x p e r im e n ta l M edicine, H u n g a r ia n A c a d e m y o f S ciences, B u d a p e s t

A K A D É M IA I K IA D Ó , B U D A PE S T 1972

@ A kadém iai K iadó, B u d a p est 1972 Printed in Hungary

F O R E W O R D

T his volum e contains th e m a te ria l o f th e S ym posium held a t th e 1971 M eeting o f th e E u ro p e a n T issue C u ltu re Society in B u d ap est. H u n g a ria n histologists were g re a tly h o noured b y being able to welcom e th is society of g re a t re p u ta tio n in H u n g a ry . T he S ym posium offered a v alu ab le occasion for p re sen tin g re p o rts on th e tissu e c u ltu re research going on in o u r co u n try a n d for y oung H u n g a ria n research w orkers to get a glim pse of c u rre n t in te rn a ­ tio n a l problem s o f tissu e c u ltiv a tio n .

E x p e rts o f tissue cu ltu re m ethods were in v ite d for th e first tim e to B u d a p e st on th e occasion o f th e In te rn a tio n a l Congress o f Zoology in 1927.

T he first in te rn a tio n a l m eeting o f e x p e rim en tal cytology w as also p a r t of th is congress. T he tissue c u ltu re tech n iq u e w hich h a d alre ad y yielded v alu ab le re su lts was a d o p te d for th e first tim e here in B u d a p e st b y in te rn a ­ tio n a l re p re se n ta tiv e s of th e biological sciences. T issue c u ltu re is to d a y a w idely ac cep ted im p o rta n t m ethod, th e significance of w hich is duely reflected b y th e p re se n t S ym posium .

W e w ould like to express o u r g ra titu d e for th e su p p o rt g ra n te d b y th e H u n g a ria n A cadem y o f Sciences enabling us to organize th e 1971 M eeting o f th e E u ro p e a n T issue C u ltu re Society a n d to p re se n t th e m a te ria l o f th e S ym posium in th is volum e.

O ur h e a rtie st th a n k s are due to all researchers a n d organizers w ho h av e g re a tly c o n trib u te d to th e success o f th e S ym posium . W e are also in d e b te d to th e P ublish in g H ouse of th e H u n g a ria n A cadem y o f Sciences fo r th e p re se n ta tio n o f th e proceedings.

T he Editors

L IS T OF P A R T IC IP A N T S

Ár o s, B.

D e p a rtm e n t o f H istology, Sem m elweis M edical U n iv ersity , B u d ap est IX , T űzoltó u tc a 58, H u n g a ry

As t a l d i, G.

T he B lood R esearch F o u n d a tio n C enter, M unicipal H o sp ital o f T o rto n a, 15057 T o rto n a, Ita ly

Ba l á z s, A.

I n s titu te of E x p e rim e n ta l M edicine, B u d a p e st V II I, Szigony u tc a 43, H u n g a ry

BÁNÓCZY, J .

I n s titu te of O ral a n d M axillo-facial S urgery, Sem m elweis M edical U n iv ersity , B u d a p e st V I I I , M ária u tc a 52, H u n g a ry

Bé l á d i, I.

I n s titu te of M icrobiology, M edical U niversity, Szeged, D óm té r 10, H u n g a ry

Be n c z ú r, M.

D e p a rtm e n t for T ra n s p la n ta tio n Im m unology, N a tio n a l B lood C enter, B u d a p e st X I, D aróczi ú t 24, H u n g a ry

Bl a z s e k, 1.

I n s titu te o f E x p e rim e n ta l Medicine, B u d a p e st V I I I , Szigony u tc a 43, H u n g ary

Bo l l, 1.

S täd tisch es K ra n k e n h a u s N eukölln, I. In n e re A bteilung, R ud o w er S trasse 56, 1 B erlin 47, B erlin-W est

Bu f f e, D .

I n s ti tu t de R echerches Scientifiques s u r le C ancer, 16 A venue-V aillant- C outurier, V illejuif (Seine), F ran c e

Bu k u l y a, B.

I n s titu te of E x p e rim e n ta l M edicine, B u d a p e st V I I I , Szigony u tc a 43, H u n g a ry

Ch è v r e m o n t, M. J . J .

I n s titu t d ’H istologie e t d ’E m bryologie, F a c u lté de M édecine, U n iv ersité de Liège, 20 ru e de P itte u rs , 4000 Liège, B elgium

Cs a b a, Gy.

D e p a rtm e n t o f H istology, Semmelweis M edical U n iv ersity , B u d a p e st IX , T űzoltó u tc a 58, H u n g a ry

DALLMAN, L.

I n s titu te of G enetics, Jó z s e f A ttila U n iv ersity , Szeged, A d y té r, H u n g a ry

In s titu te of E x p e rim e n ta l M edicine, B u d ap est V I I I , Szigony u tc a 43, H u n g a ry

Fj e l d e, A.

R osw ell P a rk M em orial In s titu te , Buffalo, New Y ork 14203, IL S .A.

Fr a n k s, L. M.

D e p a rtm e n t o f C ellular P ath o lo g y , Im p eria l C ancer R esearch F u n d , Lincoln’s In n F ields, London, WC2A 3P X , U n ite d K ingdom

Ga il l a r d, P. J .

L ab o rato riu m voor Celbiologie en H istologie, R ijnsb u rg erw eg 10, Leiden, T h e N e th e rlan d s

Ga zsó, L.

In s titu te o f N eurosurgery, B u d a p e st X IV , A m erikai ú t 57, H u n g a ry Gr o s s, W . 0 .

I n s titu t d ’H istologie, U n iv ersité de L ausanne, 9 ru e d u B ugnon, CH 1005 L ausanne, S w itzerland

Gy é v a i, A.

I n s titu te o f E x p e rim e n ta l M edicine, B u d a p e st V III, Szigony u tc a 43, H u n g a ry

Hi l w i g, I.

F arb w e rk e H oech st AG, G ew ebezüchtung H . 811, Jo h an n e salle e 10, 623 F ra n k fu rt/M a in -H ö c h s t, G erm an F e d e ra l R epublic

Ho l e c k o v á, E.

In s titu te o f P hysiology, C zechoslovak A cadem y o f Sciences, P ra h a 4, K rc , B u d ejo v ick á 1083, C zechoslovakia

Ho r v á t h, L.

N a tio n a l I n s titu te o f P u b lic H e alth , B u d ap est IX , G y á liú t 2, H u n g a ry JORDANOV, J .

I n s titu te o f M orphology, B u lg arian A cadem y of Sciences, Boul. L enin 75, bl. 103, Sofia, B u lg aria

Ka h r i, A.

2nd D e p a rtm e n t o f P ath o lo g y , U n iv e rsity of H elsinki, H a rtm a n in k a tu 3, H elsinki, F in la n d

Ka p a, E .

D e p a rtm e n t o f H istology, Semmelweis M edical U n iv ersity , B u d ap est IX , T űzoltó u tc a 58, H u n g a ry

Ke l l e r m a y e r, M.

C en tral Clinical L a b o ra to ry , M edical U n iv ersity , Pécs, Ifjú sá g ú tja 31, H u n g a ry

Ki e l e r, J .

F ib ig e rL a b o ra to ry , L u n d to ftev e j 5, K gs. L yngby, C openhagen, D en m ark K iss , J .

D e p a rtm e n t o f H istology, Semm elweis Medical U n iv ersity , B udapest IX , T űzoltó u tc a 58, H u n g a ry

Kis z e l y, Gy.

Biological I n s titu te o f M edical U n iv ersity , Szeged, K o ssu th L. sgt 35, H u n g a ry

10

К LEIN, J . С.

R adiobiological In s titu te TN O , Lange K leiw eg 151, R ijsw ijk Z. H ., T he N e th e rlan d s

Ko r it s á n s z k y, S.

D e p a rtm e n t o f H istology, Sem melweis M edical U n iv ersity , B udapest IX , T űzoltó u tc a 58, H u n g a ry

Kosa, Zs.

N a tio n a l I n s titu te o f P u b lic H e a lth , B u d a p e st IX , G yáli ú t 2, H u n g a ry Ko v á c s, Б . I.

D e p a rtm e n t o f E v o lu tio n an d G enetics, E ö tv ö s L o rán d U niversity, B u d ap est V III, M úzeum k r t 4/a, H u n g a ry

Lu n d, E.

D e p a rtm e n t o f V e te rin a ry V irology an d Im m unology, T h e R oyal V e te rin a ry a n d A g ric u ltu ra l U n iv e rsity o f C openhagen, 13, B ülow svej,

1870 C openhagen V, D en m ark Ma c ie r a-Co e l h o, A.

I n s titu t de Cancérologie e t Im m u n o g én étiq u e, 14 Av. P a u l V aillant- C outurier, 94 V illejuif, F ran c e

Ma r e e l, M.

K lin iek voor R a d io th e ra p ie en K erngeneeskunde, D e P in te la a n 115, G ent, Belgium

Ma r ó t i, M.

D e p a rtm e n t o f P la n t P hysiology, E ö tv ö s L o rá n d U niv ersity , B u d a p e st V II I, M úzeum k r t 4/a, H u n g a ry

Ma t a g n e-Dh o o s s c h e, E.

I n s titu t d ’A natom ie, L ab o rato ire d ’H istologie, ru e de P itte u rs , 4000 Liège, B elgium

MlCHL, J .

I n s titu te of P hysiology, C zechoslovak A cadem y of Sciences, P ra h a 4, K rc , B u d éjo v ick á 1083, C zechoslovakia

Ne m e s k é r i, A.

D e p a rtm e n t o f H istology, Semm elweis M edical U n iv e rsity , B u d a p e st IX , T ű zo ltó u tc a 58, H u n g a ry

Ne u p e r t, G.

P athologisches I n s titu t d er F ried rich -S ch iller-U n iv ersität, Z iegelm üh­

lenw eg 1, 69 Je n a , G erm an D em ocratic R epublic Ol á h, E.

R esearch In s titu te o f O ncopathology, B u d ap est X I I , R á th G yörgy u tc a 7, H ungary

Ol iv o, O. M.

I s titu to di A natóm ia, V ia Irn erio , 48, 40126 Bologna, I ta ly PÁLYI, I.

R esearch In s titu te o f O ncopathology, B u d a p e st X I I , R á th G yörgy u tc a 7, H u n g a ry

Pi e c k, A. C. M.

L a b o ra to ry o f C hem ical C ytology, D riehuizerw eg 200, N ijm egen, T he N e th e rlan d s

Ra p p a y, Gy.

I n s titu te o f E x p e rim e n ta l M edicine, B u d a p e st V III, Szigony u tc a 43, H u n g a ry

L ab o rato ire d ’A natom ie P ath o lo g iq u e, In serm U 77, G roupe H o sp italier N e c k e r-E n fa n ts M alades, 149, ru e le Sèvres, P a ris 15e, F rance

Rö h l ic h, P .

1st C en tral L a b o ra to ry o f E le c tro n M icroscopy, D e p a rtm e n t of H istology, Semm elweis M edical U n iv ersity , B u d a p e st IX , T űzoltó u tc a 58, H u n g a ry

Ru z ic s k a, P .

N a tio n a l I n s titu te o f P ublic H e a lth , B u d a p e st IX , G yáli ú t 2, H u n g a ry Sc h e r f t, J . P.

L a b o ra to ry for Cell Biology a n d H istology, U n iv e rsity o f Leiden, A cadem ic H o sp ital, R ijnsb u rg erw eg 10, Leiden, T he N e th e rlan d s Sc h l e ic h, A.

I n s titu t fü r E x p erim en telle K rebsfo rsch u n g d er U n iv e rs itä t H eidelberg, V oss-Str. 3, 69 H eidelberg, G erm an F ed eral R epublic

SCHWÖBEL, W .

B u n d esfo rsc h u n g san stalt fü r V iru sk ra n k h eiten d er Tiere, P au l-E h rlich - S trasse 28, 74 T übingen, G erm an F e d e ra l R epublic

Se l l y é i, M.

R ó b e rt K á ro ly H o sp ital, D e p a rtm e n t o f P ath o lo g y , B u d a p e st X I I I ., R ó b e rt K á ro ly k r t 82, H u n g a ry

Sp u r n á, V.

In s titu te o f B iophysics, C zechoslovak A cadem y of Sciences, B rn o 12, K rá lo v o p o lsk á 135, C zechoslovakia

St a r k, E .

In s titu te o f E x p e rim e n ta l M edicine, B u d a p e st V I I I , Szigony u tc a 43, H il n g ary

St ä h e l i n, H .

Sandoz AG, 4002 B asel, S w itzerland St r e e t, H. E .

School o f Biology, U n iv e rsity of L eicester, A d rian B uilding, U n iv ersity R o ad , L eicester, L E I 7R H , U n ited K ingdom

Ta n n e b e r g e r, ST.

In s titu te fü r M edizin u n d Biologie, R ob ert-R ö ssle-K lin ik , D eutsche A kadem ie d er W issenschaften zu B erlin, L indenberg W eg 80, 1115 B erlin-B uch, G erm an D em o cratic R epublic

Té t é n y i, P .

R esearch I n s titu te o f M edicinal P la n ts , B u d a p e st X II , D ániel ú t 40, H u n g a ry

Th u s t, R.

P athologisches I n s titu t d er M edizinischen A kadem ie, N o rd h ä u ser S tr. 74, 50 E rf u rt, G erm an D em ocratic R epublic

TlXIER-VIDAL, A.

L ab o rato ire de Biologie .Moléculaire, Collège de F ran ce, 11, P lace M arcelin-B erthelot, P a ris —Ve, F ran c e

Tö rő, I.

D e p a rtm e n t of H istology, Semm elweis M edical U n iv ersity , B u d a p e st

12

IX , T űzoltó u tc a 58, an d In s titu te of E x p e rim e n ta l M edicine, B u d a ­ p est V II I, Szigony u tc a 43, H u n g a ry

Tö r ö k, О.

D e p a rtm e n t o f H istology, Semmelweis M edical U n iv ersity , B u d ap est IX , T űzoltó u tc a 58, H u n g a ry

Ty i h á k, E.

R esearch I n s titu te o f M edicinal P la n ts, B u d ap est X I I , D ániel ú t 40, H u n g a ry

Vá g ú j f a l v i, D.

R esearch In s titu te o f M edicinal P la n ts , B u d ap est X II , D ániel ú t 40, H u n g a ry

Va l e n t i n i, A. F.

I s titu to di A n ató m ia, V ia Irn erio , 48, 40126 B ologna, I ta ly Ve r n e, C.-M . J .

I n s titu t d ’H istochim ie M édicale, F a c u lté de M édicine de P aris, 45 ru e des S aints-P ères, P a ris V Ie, F ran c e

Ve t t e r, J .

D e p a rtm e n t o f P la n t P hysiology, E ö tv ö s L o rá n d U n iv ersity , B u d a ­ p est V I I I , M úzeum k r t 4/a, H u n g a ry

Wa l l e r, M.

P athologisches I n s titu t, M a rtin -L u th e r-U n iv e rsitä t, H a lle -W itte n b e rg , L eninallee 14, 402 H alle (Saale), G erm an D em ocratic R epublic Wo l l e m a n n, M.

I n s titu te o f N eurosurgery, B u d a p e st X IV , A m erikai ú t 57, H u n g a ry

L IS T O F C O N T R IB U T O R S

Bá c s y, E .

I n s t i t u te o f E x p e rim e n ta l M edicine, B u d ap est, H u n g a ry Ba l á z s, A.

I n s titu te o f E x p e rim e n ta l M edicine, B udapest, H u n g a ry Bl a z s e k, 1.

I n s titu te of E x p e rim e n ta l Medicine, B u d ap est, H u n g a ry

B U K U L Y A , B .

I n s titu te o f E x p e rim e n ta l M edicine, B u d ap est, H u n g a ry Da v e y, M. R .

D e p a rtm e n t of B o tan y , U n iv e rsity of N o ttin g h a m , N o ttin g h a m , U n ited K ingdom

Fa z e k a s, 1.

I n s titu te of E x p e rim e n ta l M edicine, B u d ap est, H u n g a ry

Fj e l d e, A.

R osw ell P a rk M em orial In s titu te , Buffalo, N .Y ., U .S.A . Fr a n k s, L. M.

D e p a rtm e n t of C ellular P ath o lo g y , Im p e ria l C ancer R esearch F u n d , L ondon, U n ite d K ingdom

Gr o s s, W . O.

H istologisch-E m bryologisches I n s titu t d e r U n iv e rs itä t L a u s a n n e , L au san n e, S w itzerland

Gy é v a i, A .

I n s titu te of E x p e rim e n ta l M edicine, B u d ap est, H u n g a ry Jo b s t, К .

C en tral Clinical L a b o ra to ry , M edical U n iv ersity , Pécs, H u n g a ry Ka h r i, A. I.

2nd D e p a rtm e n t o f P ath o lo g y , U n iv e rsity of H elsinki, H elsinki, F in la n d Ke l l e r m a y e r, M.

C en tral Clinical L a b o ra to ry , M edical U n iv ersity , Pécs, H u n g a ry Ly y t ik a l n e n, A.

U n iv e rsity o f H elsinki, H elsinki, F in la n d Mi h á l y, К .

I n s titu te o f E x p e rim e n ta l M edicine, B u d ap est, H u n g a ry Ök r ö s, I.

I n s titu te of E x p e rim e n ta l M edicine, B u d ap est, H u n g a ry Pe s o n e n, S.

U n iv e rsity of H elsinki, H elsinki, F in la n d Ra p p a y, Gy.

I n s titu te o f E x p e rim e n ta l M edicine, B udapest, H u n g a ry

U n iv e rsity o f H elsinki, H elsinki, F in la n d St a r k, E.

I n s titu te o f E x p e rim e n ta l M edicine, B u d ap est, H u n g a ry St r e e t, H . E .

B o tan ical L ab o rato ries, U n iv e rsity o f L eicester, L eicester, U n ite d K ingdom

Su t t o n- Jo n e s, B.

School o f Biological Sciences, U n iv e rsity o f E a s t A nglia, N orw ich, U n ite d K in g d o m

Sz a la y, K .

In s titu te o f E x p e rim e n ta l M edicine, B u d ap est, H u n g a ry Ti x i e r-Vi d a l, A.

L a b o ra to ire de Biologie M oléculaire, Collège de F ran c e, P a ris, F ran c e TÖEŐ, I.

D e p a rtm e n t o f H istology an d E m bryology, Sem m elweis M edical U n iv ersity , B u d ap est, H u n g a ry

Tö r ö k, О.

D e p a rtm e n t o f H istology a n d E m bryology, Sem m elweis M edical U n iv e rsity , B u d ap est, H u n g a ry

16

C O N TEN T S

I . TÖRŐ

W e lc o m in g a d d re ss 19

I . Tö r ő, I . Fa z e k a s, I . Ök r ö s, Б . Bá c s y a n d Gy. Ra p p a y

F in e - s tr u c tu r a l d if fe re n tia tio n o f ly m p h o re tic u la r cells in c u ltu re 21 L . M. Fr a n k s

T h e u lt r a s t r u c t u r e o f tissu e c u ltu re cells 3]

A. Fj e l d e

T h e e s ta b lis h m e n t in c u ltu re o f p e r m a n e n t lin es o f h a e m a to p o ie tic cells, a n d r e c e n t v ir u s s tu d ie s d o n e w ith h a e m a to p o ie tic cells 37 A . T l X I E R - V I D A L

U ltr a s tr u c tu r a l fe a tu r e s o f g o n a d o tr o p ic p it u it a r y c e lls in c u ltu r e 43 A . I . Ka h r i, A. Ly y t i k ä i n e n, S. Pe s o n e n a n d A . Satire

C o m p a riso n o f th e effe c ts o f A C T H on th e n u m b e r o f m ito c h o n d r ia l p ro file s in c o rtic a l cells a n d o n th e c o n v e rs io n o f p ro g e s te ro n e -4 -14C in to c o rtic o s te ­ ro n e a n d 18-O H -D O C in tissu e c u ltu re s o f fo e ta l r a t a d re n a ls 69 A . Gy é v a i, В . Bu k u l y a, K . Mi h á l y, К . Sz a l a y a n d E . St a r k

F in e s tr u c tu r e a n d h o rm o n a l a c ti v it y o f in t a c t a n d c u ltu re d e m b ry o n ic a d ­

r e n a l cells o f d iffe re n t sp ecies 73

B . Bu k t jl y a, I . Ök r ö s, a n d E . St a r k

T h e fine s t r u c tu r e o f e m b ry o n ic r a t a d re n a ls c u ltu re d in vivo 89 I . Bl a z s e k, A. Gy é v a i, a n d A . Ba l á z s

E le c tro n -m ic ro s c o p ic s tu d y o f s p o n ta n e o u s tr a n s f o r m a tio n o f ly m p h o c y te s

a n d m o n o c y te s in tissu e c u ltu re 97

W . 0 . Gr o ss

D ie u lt r a s t r u k t u r e ll e U n te rs u c h u n g v o n Z ellen n a c h ih re r L e b e n d b e o b a c h tu n g

in d e r K u l t u r ]07

P . Rö h l ic h a n d 0 . Tö r ö k

F in e s t r u c tu r e o f th e r e t in a l p ig m e n t e p ith e liu m in o rg a n c u ltu re 127 M. Ke l l e r m a y e r a n d K . Jo b st

T h e u l t r a s t r u c t u r e o f D N P sy s te m s in tissu e c u ltu re s a s re v e a le d b y th e p o la r i­

z a tio n m ic ro sc o p e 137

H . E . St r e e t, M. R . Da v e y a n d В . Su t t o n-Jo n e s

U ltr a s tr u c tu r e o f p la n t cells g ro w in g in su s p e n sio n c u ltu re 145

S y m p . B iol. H ung., 14, p. 19 (1972) W ELC O M IN G A D D R E S S

by I . Tö r ő

PRESIDENT OF THE YEAR OF THE EUROPEAN TISSUE CULTURE SOCIETY

L adies a n d G entlem en,

I am gratified b y th e ho n o u r to be able to welcome you in B u d a p e st a t th e 1971 Session o f th e E u ro p e an Tissue C ultu re Society. P le a s a n t m em o­

ries o f bygone m eetings com e to m y m ind a n d o f th e d elig h tfu l day s we sp e n t in vario u s countries w here we assem bled.

I n th e succession o f th ese m eetings th e progress o f tissu e cu ltu re, its ev o lu tio n as a b ra n c h o f science is well reflected. A t th e o u ts e t th e m ain o b ject w as th e o b serv atio n a n d s tu d y o f th e b eh a v io u r o f e x p la n te d tissues a n d cells, b u t la te r m orphological a n d s tru c tu ra l changes a n d also differences in cell p o te n tia ls were exten siv ely in v estig ated . B y stu d y in g th e p rerequisites o f cell d ifferen tiatio n a n d d ed ifferen tiatio n several v alu ab le resu lts h av e been o b ta in e d th ro w in g light on th e o rg a n isa to ry forces m anifesting in tissues rem o v ed from th e influence o f th e organism a n d p laced in to changed circum stances. W h a t changes are m an ifested in vitro, w hich refer to d if­

ferences observed in vivo a n d w hich are th e histological a n d cytological p h en o m en a in cultures w hich b y absence rev eal th e o rg a n isa to ry forces of th e organism . P u re hom ogeneous cultures a n d th e ir c o n fro n tatio n have cleared th e w ay to th e u n d e rsta n d in g of such o rg a n isa to ry forces w hich are based on th e m u tu a l influences of different tissues a n d could give in fo rm a­

tio n for th e analysis o f ea rly em b ry o n al d ev e lo p m e n t.

I t has been observed, nam ely, t h a t ex tra c e llu la r fa cto rs d o m in atin g in cu ltu res cause in tra c e llu la r a lte ra tio n s w hich, in tu rn , affect th e h o m eo sta­

sis of th e tissues. These observ atio n s have ra ise d th e idea t h a t cell p ro lifera­

tio n w hich d o m in ates in in vitro processes a n d co n c o m itan t nucleic acid sy n th esis will in h ib it th e synth esis o f specific protein s, i.e. d ed ifferen tiatio n w ill b rin g a change also in cell p o ten tials. T he e x te n t to w hich d ed iffere n tia­

tio n o f th e cell o f higher anim als m ay serve to p rovide p re cu rso r cells is n o t know n. I t has generally been assum ed th a t genom e repression in d ed if­

fe re n tia te d cells o f v e rte b ra te s is irreversible, how ever, such repression in hig h ly d iffe ren tiated cells can be a b a te d u n d e r ce rtain ex p e rim e n ta l condi­

tions. T he s ta te of genom e repression in higher anim als as well as in sim ple organism s, depends on en v iro n m e n ta l conditions b o th w ith in a n d outside th e cell. D ifferen tiatio n can be rev ersed u n d e r a p p ro p ria te circum stances.

W e surm ise t h a t genom es su ppressed d u rin g d iffe ren tiatio n can g et released d u rin g c u ltiv a tio n a n d to g e th e r w ith th e s tru c tu ra l changes le t th e p lu rip o ten ce o f cells becom e m anifested.

F o r th is reason we believe it w ould n o t be w ith o u t m e rit to dev o te th e first d a y o f o u r C onference to th e resu lts o f u ltra s tru c tu r a l in v estig atio n s o f som e cultures.

S y m p . B iol. H ung. 14, pp. 21-29 (1972) F I N E -ST R U C T U R A L D l F F E R E N T IA T I ON

OF L Y M P H O R E T IC U L A R C ELLS IN C U L T U R E b y

I. TÖEŐ, I. Fa z e k a s, I. Ök r ö s, E. Bá c s y, and G y. Ra p p a y INSTITUTE OF EXPERIMENTAL MEDICINE, BUDAPEST, HUNGARY

T h e ch anged a ttitu d e p revailing in re c e n t y ea rs in th e research on cells c u ltiv a te d in vitro m ay be explained b y tw o reasons. One is t h a t following th e early ex p e rim en tal phase o f tissue cu ltu re tech n iq u es it is to d a y a t t e m p t ­ ed to define consciously th e in te rre la tio n b etw een e x p la n t a n d m edium , i.e.

n o t only to keep th e e x p la n t alive for a sh o rte r or longer perio d b u t also to m ake th e in vitro c u ltiv a te d cells fu n c tio n n o rm ally b y choosing th e e n v iro n m e n t properly. T he stu d ies aim ed a t th e d e m o n stratio n o f th e fu n c ­ tio n o f endocrine cells in vitro are a m ost im pressive ex am p le o f th is tre n d (S tark et ah, 1965a, b; K ow al, 1970; S ato e t al., 1970).On th e o th e r h an d , m odern m ore effective m ethods like electrophysiology, au to ra d io g rap h y , electro n m icroscopy, etc. have becom e available for s tu d y in g s tru c tu ra l changes in th e cells d u rin g c u ltiv a tio n for sh o rte r o r longer periods. This enables us to com pare th e resu lts o f cytophysiologv a n d cytom orphology a n d to draw conclusions o f m olecular-biological im po rtan ce. A lthough th e s tu d y o f th e fine s tru c tu re o f cells c u ltiv a te d in vitro has gained ground, in m ost o f th e cases th e findings have n o t been com pared w ith th o se gain ed in vivo. Such a com parison m ig h t su p p ly answ ers to fu n d a m e n ta l cvto-

TABLE 1 Cell types present in vivow

Thymocytes Reticulum cells Other cells

large6 epithelial cytoreticulum 01 fibrocytes03

m edium 0 1 I m esen ch ym al02 m ast cells

small" f f 1 granulocytes

reticular hypertrophic m acrophages plasm a cells ( ? )

m ain ly m ainly endoth elial cells01

in th e in the

cortex m edulla special ■

h istio cy tic cell

m an y m ito tic (van H aelst)

figures no m itosis no m itosis

Cell types present in vitro after 4-day culturing epithelial-like cells01 )

fibroblast-like cells0 i m an y o f them m itotic true fibroblasts03

m acrophages02^

* Symbols a and b indicate possible transformations.

com pared w ith th e original cells, a n d if so, to w h a t e x te n t; w hich cytological c h a ra c te ristic s d isap p ea r a n d w hich p ersist d u rin g cu ltiv atio n ? T he m ost im p o rta n t q u estio n p ro b a b ly is how an y such change will influence th e orig in al specific fu n c tio n o f th e cells.

T h e sam e questions arose in th e course o f o u r fin e-stru ctu ral a n d enzym e- cvtoch em ical analysis o f th y m u s expian ts.

T h e th y m u s is rich in th y m o cy tes a n d reticu lu m cells an d contains v ario u s o th e r cells, to o (Table 1). T he re ticu lu m cells are o f m esenchym al or e n to d e rm a l origin. Those deriv ed from th e e n to d erm form a pecu liar n etw o rk , th e cy to reticu lu m . T hey are easily recognizable by th e ir desm o-

F ig. 1. P o r tio n o f a n e p ith e lia l r e tic u la r cell fro m in t a c t r a t th y m u s . T o n o fila m e n ts (T), p r i m a r y ly so so m es (L ), c r is ta te m ito c h o n d r ia (M), ro u g h -s u rfa c e d e n d o p la s m ic r e t i ­ c u lu m (E R ), g ra n u le s o f d iffe re n t d e n s ity in th e c y to p la s m . X 44,500. I n s e t: A ty p ic a l d e sm o so m e c h a ra c te ris tic o f th e e p ith e lia l r e tic u la r cells. (G lu ta ra ld e h y d e -o s m iu m

fix a tio n ; u r a n y l a c e ta te an d lead c it r a t e sta in in g ) X 32,700

22

som es, tonofilam en ts a n d special vacuoles (P ig .l). T h eir specific fu n c tio n is to form th e su p p o rtin g netw ork, fu rth e r endocytosis and, in o u r an d o th e r a u th o rs ’ opinion, th e p ro d u c tio n o f th e ly m p h o c y te -stim u la tin g factor(s) (Clark, 1966, 1968; Ito , 1966; G ad a n d C lark, 1968; H iro k aw a, 1969:

G oldstein a n d W hite, 1970; G oldstein et ah, 1970; R a p p a y e t ah, 1971).

T h e m ost im p o rta n t sign o f th is la st-n a m e d fu n c tio n is th e presence of special vacoules in th e cytoplasm , e m p ty or densely packed.

R a t th y m u s frag m en ts w ere e x p la n te d on to p o f co a g u la ted chicken p lasm a a n d chick-em bryo juice on cover-slips. E a c h slide, w ith 5 frag m en ts on it, was th e n p lace d in to a L eig h to n tu b e , in w hich it w as allow ed to s ta n d for 24 h. A fte r th is tim e, 2 m l of a m edium consisting of an 8 : 2 m ix tu re o f TCM 199 a n d h e a t-in a c tiv a te d calf serum , w ith 200 I.U . of penicillin a d d e d to i t p e r ml, w as p o u re d in to each tu b e. T he cu ltu re m edium w as ex changed every o th e r day. C ultures w ere fixed a t different tim e in terv a ls in th e perio d b etw een th e 4 th a n d th e 30th d a y a fte r e x p la n ta tio n . C ultures to g e th e r w ith th e glass slides w ere fixed for electron m icroscopy in 4-5 p er

■■cent g lu ta ra ld e h y d e d ilu te d w ith 3-13 м cacodylate buffer a t p H 7 for periods o f 30 to 120 m in, a n d th e n w ashed w ith th e d ilu e n t buffer for 1 to 16 h d epending on th e co n c en tratio n o f th e fix ativ e a n d th e len g th o f fix a­

tio n tim e. S u bsequently, th e cu ltu res w ere e ith e r p ostfixed for 30 to 90 m in w ith 1 p e r ce n t osm ium te tro x id e dissolved in caco d y late buffer a n d em b ed d ed in D u rc u p an ACM, b y using p ropylene oxide, or h istochem ically an a ly se d (T able 2).

TA B L E 2

Cytochemical reactions for studying thym us expiants Cryostat sections and cultures Tests for

LM EM

Acid phosphatase N aphtol AS-BI or TR

phosphate

Sodium-/?-glycerophosphate F resh ly diazotized 5-chloro-

2-toluidine

L ead n itra te Michaelis veronal-acetate

buffer, pH 5.0

0.05 M a cetate buffer, p H 5.2 Incubation tim e: 30-60 m in In cu b atio n tim e : 1 h

Non-specific esterase N ap h to l AS-D acetate Thioacetic acid

F a st Blue В В Lead n itra te

0-1 м tris-m aleate buffer, 0-05 m cacodylate buffer,

p H 7-1 p H 5 6

In cu b atio n tim e: 2-5 m in In cu b atio n tim e: 30-60 min ( + 4 ° C )

In th e gro w th zone o f th e th y m u s e x p ia n ts (Fig. 2) th e desm osom es a n d tonofilam ents help to id en tify th e ep ith elial re tic u lu m cells even a fte r 21 days o f c u ltiv a tio n (Fig. 3). T he special vacuoles p re su m a b ly invo lv ed in th e p ro d u c tio n o f th e ly m p h o cy te-stim u la tin g factor(s) w ere n o t seen e ith e r early or la te r d u rin g c u ltiv a tio n (4-30 days), suggesting t h a t th y m ic ep ith elial re tic u la r cells do n o t prod u ce ly m p h o cy te-stim u la tin g horm one in vitro. C onsidering t h a t no th y m o cy tes w ere p re se n t in th ese cultures, it seems t h a t th e presence o f th ese cells is a p re co n d itio n for th e p ro d u c tio n

F ig . 2. S ch em e o f th e c u ltu re m e th o d a n d o f th e p r e p a r a tio n o f u l t r a t h in se c tio n s

F ig . 3. P o r tio n s o f tw o e p ith e lia l re tic u la r cells fro m a 2 1 -d ay -o ld c u ltu r e w ith a d e s ­ m o so m e (D) a n d to n o fila m e n ts (T). T h e c y to p la s m s a re r e la tiv e ly p o o r in o rg an elles.

(G lu ta ra ld e h y d e -o s m iu m fix a tio n ; u r a n y l a c e ta te a n d lead c it r a t e sta in in g ) x 34,000

o f th e ly m p h o cy te-stim u la tin g factor(s). C onsequently, one o f th e assum ed specific fu n tio n s o f ep ith elial re tic u la r cells is n o t realized in vitro.

E n d o cy to sis is a specific fu n c tio n of th e epithelial, a n d also o f th e m esen­

chym al re ticu lu m cells in th e in ta c t th y m u s ; it is co n n ected w ith th e lyso­

som es p re sen t in th e cytoplasm . I n th e in ta c t th y m u s th e re tic u lu m cells o f different origin can b e d iffe ren tiated on th e basis o f th e ir enzym e a c tiv ity

24

F ig . 4. A cid p h o s p h a ta s e (a) a n d n on-specific e s te ra s e (6) a c ti v it y in e p ith e lia l r e t i ­ c u la r cells, (a) 2 1 -d ay -o ld c u ltu re . N o te r e a c tio n p r o d u c t (H P ) in la rg e lysosom es.

T h e p o r tio n o f th e c y to p la s m is ric h in to n o fila m e n ts (T) a n d in o th e r cell o rg an elles.

X 44,000. (ft) 15-day-old c u ltu re . R e a c tio n p r o d u c t (R P ) is o b se rv a b le in d e n se b o d ie s (lysosom es). (G lu ta ra ld e h y d e -o s m iu m fix a tio n ; u r a n y l a c e ta te sta in in g ) X 27,400 (Ökrös et al., 1969a, b). T he lysosom es o f th e ep ith elial re tic u lu m cells show a m o d erate acid p h o sp h a ta se ac tiv ity , a n d a considerably low er n o n ­ specific esterase a c tiv ity (Fig. 4). I n th e m esenchym al re ticu lu m cells b o th enzym es are m ore ac tiv e th a n in th e ep ith elial cells. In th e th y m ic ex p ian ts, considerable acid p h o sp h atase a c tiv ity a n d non-specific esterase a c tiv ity were show n only b y th e m esenchym al ep ith elial cells, i.e. m acrophages (Fig. 5). T his did n o t show a n y co rrelatio n w ith th e tim e o f cu ltiv atio n . T hus th e en d o cy to tic a c tiv ity o f th e ep ith elial reticu lu m cells also seem ed to h ave decreased in o u r culture, w hereas th e m esenchym al re ticu lu m cells h ad p reserv ed th e ir intensive m acrophage function. T he fo rm a tio n

en v elo p e , e n d o p la s m ic re tic u lu m a n d som e m ito c h o n d ria . (G lu ta ra ld e h y d e -o s m iu m fix a tio n ; u r a n y l a c e ta te a n d le a d c it r a t e sta in in g ) x 13,400

26

F ig . 6. F ib r o b la s t-lik e cells (a) a n d tr u e fib ro b la s ts (6). (a) 1 9 -d ay -o ld c u ltu re . P o r ­ tio n s o f fib ro b la s t-lik e cells c h a ra c te riz e d b y a c y to p la s m p o o r in o rg an elles. T h e re is no sign o f co llag en fib re fo rm a tio n . X 19,400. (b) 9 -d a y -o ld c u ltu re . C y to p la sm s sh o w in g w ell-d ev elo p ed a n d d ila te d e n d o p la s m ic re tic u lu m . N o te th e r e la tiv e ly d e n se m a te r ia l

in c is te rn a e o f th e e n d o p la s m ic r e tic u lu m a n d signs o f fibre fo rm a tio n . X 20,000

o f th e su p p o rtin g n etw o rk was, therefore, th e only fu n c tio n o f th e ep ith elial re tic u lu m cells th e m orphological signs of w hich could still be d e m o n stra te d in vitro.

I n vitro conditions n o t only influence th e specific fu nctions of th e cells b u t also change th e cellular com position of th e original ex p la n t. T he change in th e incidence o f different cell ty p e s is due to th e declined v ia b ility o f som e cell ty p es, to th e a lte re d p ro liferatio n ra te o f n o n -p a ren c h y m a l cells,

m ito tic cells is re la tiv e ly g re at, b u t th e ir id en tificatio n is difficult. The n u m b e r o f ep ith elial re tic u lu m cells increases parallel w ith th e tim e of c u ltiv atio n , because th ese cells regain th e ir m ito tic ab ility in cu ltu re, i.e.

th e ir a u to sy n th e tic fu n ctio n is realized in vitro. As a m a tte r o f fact, th e ir nuclei co n tain little h etero ch ro m atin , a n d one or tw o m a rk e d nucleoli.

T here is also an increase in th e n u m b e r of m acrophages. T his m ay be e x p la in ­ ed w ith th e m esenchym al re tic u lu m cells becom ing capable o f m itosis or w ith th e tra n sfo rm a tio n o f th e cells o f th e original e x p la n t into m acrophages (Nelson, 1969). A ccording to several a u th o rs, lym phocytes can be tr a n s ­ form ed in to m acrophages (Nelson, 1969); in o u r m ateria l th e in vitro tra n sfo rm a tio n o f ly m p h o cy tes in to m acrophages could n o t be p ro v e d b u t it could n o t be ex cluded either.

O u r th y m u s e x p ia n ts co n tain e d n o t only ep ith elial re ticu lu m cells and m acrophages b u t also a n u m b e r o f tru e fibi'oblasts a n d fibroblast-like cells (Fig. 6). T he signs o f in ten siv e p ro te in sy n th esis: a b u n d a n t endoplasm ic reticu lu m , th e a p p e ara n ce o f d ila te d saccules a n d cystern ae, a n d th e presence o f collagenous fibres, all p o in t to th e ir original specific fu n ctio n h av in g been p reserved. O f th e fu n ctio n s o f th e fibroblast-like cells whose cyto p lasm is po o r in organelles, only a u to re p ro d u c tio n was evident.

T he living organism as well as th e in d iv id u a l organs are con tro lled b y general a n d specific re g u la to ry system s (Goss, 1967). I n th e case o f isolated cells in vitro th e re is no such com plex regu latio n . T he re g u la to ry factors w hose presence is needed in th e artificial m edia to m a in ta in th e specific fu n c tio n of th e e x p la n te d cells for a w ell-defined period, or infinitely, could be re liab ly identified only for a lim ited n u m b e r of cells. A m ore p e n e tra tin g insight in to th e facto rs influencing th e specific fu n ctio n s a n d a u to sy n th e tic a c tiv ity o f th e cells o f ly m p h o reticu lar organs in in vitro cu ltu res w ould help considerably in th e u n d e rsta n d in g of th e re g u la to ry m echanism functioning in vivo. T his requires, in ad d itio n to fu n c tio n a l in vestigations, th e know ledge o f th e fine s tru c tu re of th e cells. F u r th e r fin e-stru ctu ral in v estig atio n s on c u ltiv a te d th y m ic cells m ig h t significantly c o n trib u te to o u r know ledge o f th e fu n c tio n a l m echanism s o f th e th y m u s.

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Cl a r k, S. L ., j r. (1968): I n c o r p o ra tio n o f s u lfa te b y th e m o u se th y m u s : its re la tio n to s e c re tio n b y m e d u lla r y e p ith e lia l cells a n d th y m ic ly m p h o p o ie sis. J . exp. M e d . 128, 927

Ga d, P . a n d Cl a r k, S. L ., j r. (1968): In v o lu tio n a n d re g e n e r a tio n o f th e th y m u s in m ice, in d u c e d b y b a c te r ia l e n d o to x in a n d s tu d ie d b y q u a n ti ta t iv e h is to lo g y a n d e le c tro n m ic ro sc o p y , A m er. J . A n a t. 122, 573

Go l d s t e i n, A . L ., As u a n u m a, Y . a n d Wh i t e, A . (1970): T h e th y m u s as a n en d o c rin e g la n d : p ro p e r tie s o f th y m o s in , a n e w th y m u s h o rm o n e . I n , As t w o o d, E . B . (E d .):

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Ne l s o n, D. S. (1969): M acrophages a nd I m m u n i t y . N o rth - H o lla n d , A m s te rd a m Ök r ö s, I ., Fa z e k a s, I ., Bá c s y, E ., Ra p p a y, Gy. a n d Törő, I . (1969a): H id ro litik u s

e n zim ek a k ti v it á s á n a k e le k tro m ik ro s z k ó p o s v iz s g á la ta in vitro te n y é s z te t t p a tk á n y th y m u s -s e jte k b e n . (E le c tro n m icro sco p ic in v e s tig a tio n o f th e a c ti v it y o f th e h y d r o ­ ly tic en z y m e s o f in vitro c u ltiv a te d r a t th y m u s cells). B io l. K ö z i. 17, 3

Ök r ö s, I ., Fa z e k a s, I ., Bá c s y, E ., Ra p p a y, Gy. a n d Tör ő, I . (19696): H y d ro litic en z y m e a c ti v it y o f r a t th y m ic cells g ro w n in vitro. A n e le c tro n m icro sco p ic s tu d y . H istochem ie 20, 108

Ra p p a y, Gy., Bu k u l y a, B . a n d Bá c s y, E . (1971): F in e s tr u c tu r e , d is tr ib u tio n a n d fu n c tio n o f th e r a t th y m ic re tic u la r cells in th e p e r i n a ta l life. A cta hiol. A ca d . S e i.

hung. 22, 187

Sa t o, G., Augttsti-Tocco, G ., Po s n e r, M. a n d Ke l l y, P . (1970): H o rm o n e -se c re tin g a n d h o rm o n e -re s p o n siv e cell c u ltu re s . I n , As t w o o d, E . B . (E d .): R ecent Progress in H orm one Research. A c a d e m ic P re s s, N ew Y o rk , 26, 539

St a r k, E ., Gy é v a i, A ., Sz a l a y, K . a n d A c s, Z s. (1965a): H y p o p h y s ia l-a d re n a l a c ti v ­ it y in co m b in e d h u m a n fo e ta l tis s u e c u ltu re s , C anad. J . P h y sio l. P harm acol. 43, 1 St a r k, E ., Gy é v a i, A ., Sz a l a y, K . a n d Pó s a l a k y, Z. (19656): S e c re tio n o f a d r e n o ­ c o rtic o tro p ic h o rm o n e b y h y p o p h y s e a l cells g ro w n in m o n o la y e r c u ltu re . J . E n d o cr.

31, 291

S y m p . Biol. H ung. 14, pp. 31-35 (1972) T H E U L T R A S T R U C T U R E O F T IS S U E C U L T U R E C ELLS

by

L . M. Fr a n k s

DEPARTMENT OF CELLULAR PATHOLOGY, IMPERIAL CANCER RESEARCH FUND, LONDON, UNITED KINGDOM

T he p roblem of id e n tity is a m a jo r p re o ccu p a tio n for philosophers in th e 2 0 th c e n tu ry an d m y ta lk to d a y concerns itse lf w ith a sm all fa cet o f th is c e n tra l problem o f o u r civilization. I t underlines th e u n c e rta in tie s o f an id e n tity w hich depends only on appearan ces, p a rtic u la rly w hen those ap p earan ces are shadow s. M orphology is a useful guide to id e n tity b u t, as in so m an y o th e r cases, it is th e p a tte r n w hich is as im p o rta n t as th e d etailed s tru c tu re o f th e in d iv id u a l com ponents. T hus m orphology will allow us to id e n tify w ith c e rta in ty m ost tissues b u t th e identification of an in d iv id u al cell from an y given tissu e m ay n o t be possible unless it has som e clearly recognisable m arker. T his is th e ce n tral problem for tho se concerned w ith th e u ltra s tru c tu r e of tissu e cu ltu re cells.

T H E U L T R A S T R U C T U R E O F M O U S E C E L L L IN E S

I am going to describe th e u ltra s tru c tu r e o f a n u m b e r of cell lines derived from n o rm al tissues a n d from tu m o u rs a n d t r y to draw som e conclusions from o u r findings. T he first ex p e rim en t I am going to describe was set up to com pare th e g ro w th p o te n tia l o f y oung a n d old tissues (F ra n k s an d H enzell, 1970). Since th e re is an increasing incidence of tu m o u rs in som e organs w ith age o u r original prem ise was th a t if th e re was a cellular basis for th is increased neo p lastic p o te n tia l, cells from old organs should undergo sp o n tan eo u s n eoplastic tra n sfo rm a tio n m ore read ily th a n cells from young tissues. T h irty -six cell lines w ere estab lish ed from vario u s organs o f em bryo (18 day), young (3-20 days) a n d old (28-34 m onths) C3H a n d С57.ВТ(а‘) mice. S p o ntaneous n eoplastic change occurred in 16 of th ese lines, b u t th e re w as no specific age relatio n sh ip . T he u ltra s tru c tu re o f 11 tu m o u r lines a n d 13 n o n -tu m o u r lines w ere stu d ie d a t different tra n s fe r generations b y tran sm issio n electro n m icroscopy (F ra n k s a n d W ilson, 1970) a n d som e b y scanning electron m icroscopy (H odges. 1970). T he cells in all cu ltu res w ere sim ilar w h a te v er th e organ from w hich th e y were deriv ed (kidney, lung, blad d er, tongue, h e a rt, spleen, p ro sta te , p erito n eu m a n d spinal cord).

W e h ave described th e u ltra s tru c tu re o f th ese cells in d etail in an earlier p a p e r (F ra n k s an d W ilson, 1970). T here were 2 m ain ty p es o f cell. T y p e 1 cells h ad ro u n d e d or b ea n -sh ap e d nuclei w ith a th in rim o f m arg in al c h ro m a­

tin , a n d th e cyto p lasm co n tain e d som e rough endoplasm ic reticu lu m , m an y free ribosom es, a sm all Golgi zone a n d re la tiv e ly few lysosom es, au to p h ag ic vacuoles an d m ito ch o n d ria. Cell processes were s h o rt a n d few in num ber.

endoplasm ic reticulum . T he cells were som etim es ro u n d e d b u t m ore u su ally th e surface was v ery irre g u la r w ith long sheet-like cytoplasm ic fringes ex ten d in g for a considerable d istan ce from th e cells. In suspensions p re p a re d b y scraping th ese processes w ere o rie n te d along a lay er o f ex tra cellu lar fibrillar m ateria l a n d o ften in te rd ig ita te d w ith sim ilar processes from o th er cells. A lthough m ost cells could easily be classified as ty p e 1 or 2 th ere w ere som e a ty p ic a l cells in m ost cultures. M ost o f th ese h ad th e cytoplasm ic c h a rac te rs o f ty p e 1 cells b u t th e nuclei w ere m ore convoluted. O th e r cells h a d m ore cytoplasm ic organelles th a n th e ty p ic a l ty p e I cells. T his suggests th a t th e re m ay be a tra n s itio n betw een ty p e 1 a n d ty p e 2 cells. O ccasional g ia n t cells were also p resen t. O nly th e ir size distin g u ish ed th e m from o th e r ty p e I an d ty p e 2 cells. T here w ere also a n u m b er of o th e r u ltra s tru c tu r a l features. M ost o f th e cells h ad in tra c e llu la r actin -lik e filam ents (F ran k s e t al., 1969), som etim es in v ery large num bers. M icropinocytotic vesicles were v ery fre q u e n t in th e p erip h e ral areas of cells, especially in those w ith m an y filam ents. Specialized cell co n tac ts w ere present betw een all cell ty p e s m o stly in te rm e d ia te ju n ctio n s (zonula adherens) b u t a few aty p ic a l tig h t ju n ctio n s (zonula occludens) were also found. Thickenings o f th e in n er a sp e ct o f th e p lasm a m em brane, resem bling a tta c h m e n t bodies o f sm ooth m uscle, w ere o ften found. These w ere u su ally associated w ith e x tra cellu lar m a te ria l resem bling basal lam ina. T he d is trib u tio n of th is basal lam ina-like m a te ria l is b e st seen in p re p a ra tio n s in w hich th e cells are fixed a n d em b ed d ed in situ, w ith o u t rem oval from th e glass on w hich th e y h av e grow n. U sing a tech n iq u e developed b y m y colleague, Mr. Cooper, it can be seen t h a t th is m ateria l is p ro b a b ly also concerned w ith th e a tta c h m e n t o f cells to glass. M ost of th e o th e r ex tra c e llu la r m ateria l was n o t collagen. I t h a d 2 com ponents, an am orphous m a te ria l a n d fine tu b u la r fibrils a b o u t 100À in d iam eter, resem bling th e fibrillar com ponent o f elastic tissue.

T he surface s tru c tu re o f som e of th ese cells h a d been described b y D r.

Gisele H odges (1970). W hen th e cell lines w hich have u ndergone neoplastic tra n sfo rm a tio n are in o cu lated in to syngeneic m ice th e y all produce sim ilar tu m o u rs, w h a te v er th e org an o f orig in (F ra n k s e t al., 1970).

T H E U L T R A S T R U C T U R E O F C E L L L I N E S F R O M O T H E R S P E C IE S T hese findings ra ised tw o questions. F irstly , are th e re in fa c t 2 fixed cell ty p e s or can one ty p e change in to th e o th e r? Secondly, w here do th e tissue c u ltu re cells come from ? To answ er th e first questio n we estab lish ed 8 clones from one cell line. Tw o o f these co n tain e d b o th cell ty p es, one contained ty p e 2 cells only a n d 5 co n tain e d only ty p e 1 cells. T he tw o m ixed ‘clones’

m ay n o t h ave been tru ly cloned so th a t it seems t h a t tw o ty p e s m ay be sta b le b u t we c a n n o t y e t exclude th e p ossibility o f a tra n sitio n . We hope to answ er th is questio n sh o rtly .

32

T he second q u estio n is m ore difficult to answ er. One p ossibility is t h a t o u r c u ltu re conditions do n o t allow th e su rv iv al o f d iffe ren tiated p a re n c h y ­ m al cells from th e organs we h av e cu ltu re d a n d th a t we h av e selected out th e sam e ‘u n d iffe re n tia te d ’ cells from each organ. T he o th e r p ossibility is t h a t th e tissue cu ltu re cells are in fa ct deriv ed from specific p aren ch y m al cells b u t undergo a s tru c tu ra l m o dulation in culture, so t h a t th e y all have a sim ilar ap p earance. T here are few d etailed d escriptions o f th e u ltra s tru c tu re o f cell lines d erived from norm al tissues. We, therefore, m ade a b rief su rv ey o f a n u m b er o f cell lines estab lish ed in o th e r lab o rato ries, an d from o th e r species th a n th e mouse. These included th e B alb/c 3T3 line estab lish ed by A aronson a n d T odaro (1968), N IL cells a n d secondary h a m ste r em bryo cu ltu res (all p ro v id ed b y D r. Ia n M cPherson) an d a line derived from ra t liv er b y D r. K aig h (provided b y D r. B. W einstein). T hese cell lines h av e an u ltra s tru c tu re sim ilar to t h a t we have described in o u r m ouse cell lines.

F in a lly we have ex am in ed cells from m an y lines deriv ed in o u r lab o rato ries from h u m an em bryos (F ra n k s a n d Cooper, in p re p ara tio n ). I n lines d erived from lung, th e u ltra s tru c tu re is sim ilar to t h a t I have described, no t only in cell ty p e s b u t in th e d istrib u tio n o f ex tra -ce llu lar basal lam ina-like m aterial m ost easily seen in cells sectioned in situ.

Again, th e re are few d etailed descriptions of th e u ltra s tru c tu re of h u m an em b ry o cell lines b u t we were encouraged to find a descrip tio n o f th e u ltra s tru c tu re of cells d erived from h u m an skin cultures (Comings and O kada, 1970) which a p p e a r to be id en tical to tho se we h av e described.

T H E P O S S IB L E N A T U R E O F T IS S U E C U L T U R E C E L L S

I n p rim a ry e x p la n t cultures or in organ cultures, specific ep ith elial cells can be recognized a t th e lig h t m icroscope a n d u ltra s tru c tu ra l levels (e.g.

F ra n k s a n d B a rto n , 1960) b u t, as has been recognized for m an y years, d iffe ren tiated cells in cu ltu re are u su ally ‘o vergrow n’ b y so-called fibroblasts.

I f th e re is selection o f cells ra th e r th a n a s tru c tu ra l change affecting dif­

fe re n tia te d cells, th e cells w hich are ev e n tu a lly selected should be presen t in th e in itial cell suspensions or ex p ian ts from w hich th e cu ltu res were established. We, therefore, exam ined p rim a ry cell suspensions an d p a rtia lly trv p sin ized tissue frag m en ts to see if we could id e n tify cells sim ilar to tho se w hich ev e n tu a lly p re d o m in a te in culture.

In suspensions p re p a re d from m ouse kid n ey (F ran k s a n d W ilson, 1970) cells resem bling ty p e 1 a n d ty p e 2 cells can be seen associated w ith th e glom erular capillaries a n d a tta c h e d to th e o u te r walls o f som e tubules.

I n p re p a ra tio n s from h u m an em bryo lung th e possible site of origin of th e cells can be seen m ore clearly. T he histology o f th e h u m an em bryo lung has been described b y Cooper (1938) an d th e ap p earan ces in p rim a ry cu ltu res b y C hesterm an an d F ra n k s (1960). T he ep ith elial cells have a d istin c tiv e ap p earan ce q u ite different from t h a t o f th e cells w hich are found in th e la te r cultures. I n th e stro m a groups of m esenchym al cells can be seen. Some are fibroblasts a n d o th ers h ave m an y o f th e fe atu res seen in th e ty p e 2 tissue c u ltu re cells, i.e. irre g u la r co nvoluted nuclei, in trac ellu la r fibrils, o ften in large num bers, p erip h eral m icropinocytotic vesicles, specialized cell co n tacts, etc. M any ol tnese cells are in solid cords, b u t o th ers su rro u n d